Hello.
We are planning expression of a restriction enzyme in E. coli, and wondering how much of the corresponding methyltransferase is needed to be expressed in order to protect the host genome from the endonuclease activity. Do we need a strong promoter such as T7 or a more moderate one should be fine?
Thanks for sharing your experience.
You don't need very much. It doesn't matter how much endonuclease you make, you only need enough methyltransferase to protect all the sites in the genome. And in nature this is achieved by a single copy of the gene in the native host chromosome that is not overexpressed. So just by cloning the methyltransferase gene on a multicopy plasmid with some reasonable promoter (lacZ is fine or possibly even the native promoter depending upon the origin) will be more than sufficient.
Hi Mohsen,
It will be important you clone both genes in compatible vectors for E. coli (as pET and pACYC) or both genes inserted in the same vector as pETDuet. Thus, you will guarantee that each E. coli cell will express both proteins. Best wishes!
Michael J. Benedik
Thank you for your comment. Please correct me if I'm wrong, but lac promoter might not suit my need perfectly, as it needs activated CAP in order to be active constitutively. What about lacUV5?
Maxuel Andrade
Thanks for your comment. I would definitely consider using two vectors with different origin of replications.
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